Immune stimulatory cytokines can be exploited to treat human ailments including cancer. Amongst cytokines identified for such use, Granulocyte-Macrophage-Colony Stimulating Factor (GM-CSF) has been under much scrutiny since it acts directly on the adaptive immune system by enhancing antigen presentation as well as costimulation (Dranoff et al. 1993). Furthermore, second generation strategies linking innate and adaptive immunity using GM-CSF delivered as a fusion cytokine (fusokine) with other immune stimulatory proteins such as Interleukin-2 (IL-2) and IL-3 have been developed (Stagg et al 2004). GM-CSF was first described as a growth factor for granulocyte and macrophage progenitor cells. However, GM-CSF is also an important mediator for inflammatory reactions produced by T lymphocytes, macrophages and mast cells present at sites of inflammation (Demetri and Griffin, 1991). GM-CSF is a strong chemoattractant for neutrophils. It enhances microbicidal activity, phagocytotic activity and cytotoxicity of neutrophils and macrophages. An important feature of GM-CSF is that it greatly enhances the state of antigen presentation on dendritic cells, known to be crucial mediators of acquired immunity. The DNA and protein sequences of GM-CSF have been protected under PCT application WO8600639 and the derived patents.
CCL2, a chemokine of the CC family, was first characterized as a monocyte-chemoattracting protein. Its expression is promoted after exposure to inflammatory stimuli such as IL-1, TNF-alpha or IL-4. CCR2, the receptor for CCL2, is a Gi-coupled receptor highly expressed on monocytes, dendritic cells, T and B cells. Subsequent studies showed that CCR2 is also expressed on activated and memory T cells, including both TH1 and TH2 cells. Both CCL2- and CCR2-deficient mice show defects in monocyte recruitment. Animals that lack CCL2 show diminished T cell responses. In contrast, CCR2 deficient mice have markedly reduced T cell IFN-gamma responses, defects in clearance of intracellular pathogens and increased resistance to the TH1-mediated disease, such as experimental autoimmune encephalomyelitis (EAE).
The success of inflammatory reactions rely on the co-ordination and control of immune cell trafficking, which is mediated by chemokines, a large group of chemotactic molecules divided in four groups (Luther and Cyster, 2001). These small polypeptides usually bind to their cognate G protein-coupled receptors (GPCRs) mediating various physiological processes including inflammation, allograft rejection, autoimmunity, viral infections and lymphopoeisis (Campbell and Butcher, 2000; Lee et al. 2003; Sallusto et al. 2000; Ansari et al. 2007; Fife et al. 2000). As such, numerous approaches have been employed to modulate GPCRs activities ranging from blocking antibodies to small molecule inhibitors (Loberg et al. 2007; Onai et al. 2000; Coffield et al. 2003). Unfortunately however, most of these strategies failed or had multiple limitations such as toxicity, lack of target specificity, paracrine bystander effects or in vivo inefficacy (Onai et al. 2000; Coffield et al. 2003; Engel et al. 2000).
A novel approach for GPCR modulation could be combinatorial fusokines. In other words, the fusion of 2 different cytokine cDNAs as one open reading frame that might lead to a new compound with unanticipated pharmacological properties as previously reported (Rafei et al. 2006). Recently, it was found that a 4-amino acid (aa) truncation at the N-terminus of CCL2 (mpCCL2) leads to the generation of MCP-1 (5-76) which can completely reverse the biochemical property of the molecule from agonist to antagonist (McQuibban et al. 2002). The present inventors have previously demonstrated the inhibitory influence of mpCCL2 on both humoral and cellular pathologies by blocking the generation of inhibitory antibodies following recombinant factor VIII immunization in haemophilic hosts (Rafei et al. 2008), and for the alleviation of EAE disease score (Rafei et al. 2009 a and b).
Chemokines and their receptors are involved in normal physiological responses but they can, under certain circumstances, exacerbate pathological immune reactions (Luther et al. 2001). For example, CCR2 is largely implicated in the pathophysiology of graft-versus host disease, EAE, inflammatory bowel disease and many more pathologies (Israel et al. 2004; Cheung et al. 2008; Terwey et al. 2005; Huang et al. 2001; Uguccioni et al. 1999; Shahrara et al. 2008). As a result, GPCRs have generated considerable interest in the pharmaceutical industry as drug targets. Various difficulties, however, were encountered in generating compounds that can specifically target CCRs without side effects. For instance, the development of intrakine (Onai et al. 2000) or degrakine molecules (Coffield et al. 2003), which are specific chemokines linked to an endoplasmic reticulum (ER) retention signal sequence (KDEL) on their carboxy termini, have been shown to sequester target GPCRs in the ER to prevent their transport to the cell surface or induce their degradation. Even though efficient in preventing or reducing chemokine stimulation, these molecules were linked to intracellular toxicity not to mention their passive diffusion outside of the cell (Coffield et al. 2003). Nevertheless, transducing target cells is required for the success of this strategy, an approach that is unfeasible in the context of ubiquitous expression of target GPCR.